Carbamoylation of amino groups in peptides via N-aryloxycarbonyl intermediates

ABSTRACT

Method of synthesising peptides containing one or more amino acid residues bearing an N-carbamoyl functional group, by aminolysis of N-aryloxycarbonyl derivatives, which are excellent synthesis intermediates for the preparation of various peptides containing amino acid residues bearing a ureino group, such as citrulline, homocitrulline, 2-amino-4-ureidobutyric residues.

This application is a Continuation of application Ser. No. 08/034,705,filed Mar. 19, 1993, now abandoned.

BACKGROUND OF THE INVENTION

The present invention relates to a new method of synthesising peptidescontaining one or more amino acid residues bearing an N-carbamoylfunctional group. The invention also relates to new synthesisintermediates used in this method and to new peptide fragments.

Various synthetic peptides, used especially as reagents for analyticaltests, as sweetening agents, as synthetic hormones or as medicinalproducts, contain amino acid residues bearing an N-carbamoyl functionalgroup, either on the α-amino functional group of the N-terminal residue,or on the ω-amino functional group of a diamino acid residue, that is tosay at the end of a side chain. Such diamino acids are especiallynorcitrulline (Nci), citrulline (Cit) or homocitrulline (Hci).

The introduction of an amino acid residue bearing an N-carbamoylfunctional group into a peptide chain, starting directly with the aminoacid carrying this carbamoyl functional group, has some disadvantages.Indeed, the presence of a ureido or ureino functional group seriouslyinterferes with known methods of peptide synthesis, both in the "solid"phase based on the so-called Merrifield technique, and in the liquidphase. The chemical yields are poor and an adequate chiral purity isobtained only at the cost of laborious and costly purifications.Furthermore, in the case of diamino acids, the synthesis of amino acidsbearing a ureido or ureino functional group at the end of the sidechain, in pure enantiomorphous form, is difficult and therefore costly.

The synthesis of a peptide containing an amino acid residue bearing anN-carbamoyl functional group is therefore generally carried out in twostages. In a first step, a precursor peptide containing the amino acidresidue bearing the amino functional group to be carbamoylated issynthesised. In a second step, the amino functional group to becarbamoylated is converted, inside the peptide chain, to a ureinofunctional group. For example, it is well known to convert, bycarbamoylation by means of sodium isocyanate, the α-amino functionalgroup of the N-terminal valine residue of the β chain of sickle cellhemoglobin, to a ureido functional group. (Lehninger A. L.;Biochemistry; (1975) second Edition, p. 149)

However, this known method does not appear to be satisfactory forpeptide synthesis, mainly because of the insufficient specificity of thecyanates or isocyanates for the amino functional group(s) to becarbamoylated and because of the racemisation which may be induced bysuch a treatment.

Moreover, R. A. Boissonnas and G. Preitner (Helvetica Chimica Acta,(1953), Vol. 36 (4), p. 875-886) studied the removal ofphenyloxycarbonyl (PhOC) and p-tolyloxycarbonyl (TOC) groups, which areused as groups for blocking the α-amino functional group of some α-aminoacids, by means of various dissociating agents. All the dissociatingagents used led to the complete removal of these groups and to therelease of the α-amino functional group.

The invention overcomes the disadvantages of known methods of preparingN-carbamoyl-peptides by providing a new method for preparingN-carbamoyl-peptides with an improved chemical yield, which makes itpossible to preserve to a remarkable extent the chiral purity of thestructures used.

SUMMARY OF THE INVENTION

Consequently, the invention relates to a method of producing a peptidecontaining one or more N-carbamoyl functional groups, according to whichan intermediate peptide containing one or more aryloxycarbonyl groupsconsisting of a group of general formula ##STR1## attached to thenitrogen atom of an amino functional group and in which R3 represents anaryl group, unsubstituted or substituted by one or more alkyl groupscontaining 1 to 4 carbon atoms, is reacted with a compound of generalformula R1R2NH in which R1 and R2 represent, independently of eachother, hydrogen atoms, alkyl, cycloalkyl or aralkyl radicals, containingat most 12 carbon atoms, or in which R1 and R2 together form analicyclic radical containing 3 to 6 carbon atoms.

Peptide is understood to mean, for the purposes of the presentinvention, any natural or synthetic compound consisting of thecombination of at least two natural or synthetic amino acids, and moreparticularly combined by an amide bond. In a wider sense, the term"peptide" is also understood to include any peptide structure some ofwhose functional groups are optionally substituted by protecting groupsor activating groups.

Amino acid is understood to mean below any organic acid possessing atleast one carboxyl functional group and at least one primary orsecondary amino functional group, such as natural amino acids orsynthetic amino acids. In a wider sense, the term "amino acid" is alsounderstood to include below any amino acid some of whose functionalgroups are optionally substituted by protecting groups or activatinggroups. The amino acids are preferably those containing at least onecarbon atom carrying both a carboxyl functional group and an aminofunctional group.

N-carbamoyl functional group is understood to mean below any carbamoylfunctional group --CO--NH₂ attached to the nitrogen atom of an aminofunctional group of an amino acid or of a peptide. In a wider sense, theterm "carbamoyl" is also understood to include below any carbamoylfunctional group at least one of whose two hydrogen atoms is substitutedby an alkyl, cycloalkyl or aralkyl radical containing at most 12 carbonatoms or whose two hydrogen atoms are substituted by a bivalentalicyclic radical containing 3 to 6 carbon atoms. The carbamoyl functiongroup forms, with the nitrogen atom to which it is attached, a ureino orureido functional group depending on whether the carbamoyl functionalgroup is substituted or unsubstituted.

In the compound of general formula R1R2NH, alkyl, cycloalkyl, aralkyl oralicyclic radicals are understood to mean, for the purposes of thepresent invention, exclusively hydrocarbon radicals as well ashydrocarbon radicals substituted by functional groups comprising atleast one oxygen, sulfur or nitrogen atom, such as carboxyl, hydroxyl,sulfhydryl, indolyl and imidazolyl groups.

In the remainder of the description, just as N-carbamoyl functionalgroup is used to designate any carbamoyl functional group --CO--NH₂attached to the nitrogen atom of an amino functional group of an aminoacid or of a peptide, N-aryloxycarbonyl will be used to designate anyaryloxycarbonyl group R3--O--CO-- attached to the nitrogen atom of anamino functional group of an amino acid or of a peptide.

In the method according to the invention, the nature of R3 is critical.Indeed, it emerged, surprisingly, that unlike the conventional groupsfor protecting an amino functional group, such as for example theN-benzyloxycarbonyl group or the N-phthaloyl group, which groups, in thepresence of ammonia or an amine, release the amino functional groupunaltered, the N-aryloxycarbonyl groups lead, in the presence of suchcompounds, to the formation of an N-carbamoyl functional group.Generally, R3 is a group containing at most 12 carbon atoms. Most often,R3 is a phenyl, naphthyl, tolyl, xylyl, mesitylyl, ethylphenyl,diethylphenyl, propylphenyl or isopropylphenyl group. Preferably, R3 isa phenyl or tolyl group. In a particularly preferred manner, R3 is aphenyl or p-tolyl group. In this latter case, the intermediate peptideused in the method according to the invention is theN-phenyloxycarbonyl(PhOC) derivative or the N-tolyloxycarbonyl (TOC)derivative respectively. By analogy with the names PhOC and TOC, thevarious aryloxycarbonyl groups which can be used in the method accordingto the invention are designated below by the generic abbreviation ArOC.

Excellent results have been obtained in the method according to theinvention with R1R2NH compounds for which R1 and R2 radicals arehydrogen atoms or exclusively hydrocarbon radicals. Such compounds are,for example, ammonia or the primary or secondary amines chosen fromalkylamines, cycloalkylamines, aralkylamines and heterocyclic amines.These amines generally contain at most 12 carbon atoms. By way ofnonlimitative examples, there may be mentioned, as alkylamines,methylamine, ethylamine, n-propylamine, isopropylamine, n-butylamine,tert-butylamine, 2-methylbutylamine, dimethylamine, diethylamine andN-methyl-N-ethylamine, as cycloalkylamines, cyclopentylamine andcyclohexylamine, as aralkylamines, benzylamine, N-(phenylethyl)amine,N-(phenylpropyl)amine and N-(phenylbutyl)amine, as heterocyclic amines,pyrrolidine and piperidine.

By way of illustration of R1R2NH compounds for which R1 and/or R2radicals are hydrocarbon radicals substituted by functional groupscomprising at least one oxygen, sulfur or nitrogen atom, there may bementioned the amino acids, as defined above.

In the case where the compound R1R2NH is ammonia, the N-carbamoylfunctional group formed by the method according to the invention isunsubstituted and the peptide obtained therefore contains a ureidofunctional group. In all the other cases (R1 and/or R2 different fromH), the N-carbamoyl functional group formed by the method according tothe invention is substituted by at least one group containing one ormore carbon atoms and the peptide obtained thus contains a ureinofunctional group.

In a peptide, 2 types of amino functional groups are capable of beingcarbamoylated: on the one hand, the α-amino functional group of theN-terminal residue and, on the other hand, the ω-amino functional groupof a diamino acid residue regardless of the position of this diaminoacid within the peptide chain.

The method according to the invention makes it possible to prepareN.sup.ω -carbamoyl-peptides and N.sup.α -carbamoyl-peptides. N.sup.α-carbamoyl-peptide is understood to mean any peptide bearing a carbamoylfunctional group, as defined above, on the α-amino functional group ofthe N-terminal residue of a peptide chain. N.sup.ω -carbamoyl-peptide isunderstood to mean any peptide bearing a carbamoyl functional group, asdefined above, on one or more ω-amino functional groups of any diaminoacid residue situated in any position in the peptide chain.

The method according to the invention appears to be particularlyefficient for the production of N.sup.ω -carbamoyl-peptides startingwith an intermediate peptide bearing an aryloxycarbonyl group on one ormore ω-amino functional groups. Preferably, the method according to theinvention is used for converting, inside a peptide structure, a lysine,ornithine or 2,4-diamino-butyric residue, whose ω-amino functional groupat the end of a side chain is substituted by an ArOC group, to ahomocitrulline, citrulline or 2-amino-4-ureido-butyric (norcitrulline)residue respectively, regardless of their configuration (D, L or DL).

The intermediate peptide, containing one or more N-aryloxycarbonylgroups, which is used in the method according to the invention mayadditionally contain other functional groups substituted by protectinggroups or activating groups.

Protecting group or activating group are understood to mean any compoundcited in the literature for this purpose. By way of illustration of suchprotecting or activating groups capable of substituting one or morefunctional groups of the intermediate peptide containing one or moreN-aryloxycarbonyl groups used in the method according to the invention,there may be mentioned:

a) as groups protecting the amino functional group,

substituted or unsubstituted groups of the alkyl or aralkyl type,containing heteroatoms or otherwise, such as benzyl, diphenylmethyl,di(methoxyphenyl)methyl and triphenylmethyl (trityl) groups,

substituted or unsubstituted groups of the acyl type such as especiallyformyl, acetyl, trifluoroacetyl, benzoyl and phthaloyl groups,

substituted or unsubstituted groups of the aralkyloxycarbonyl type suchas especially benzyloxycarbonyl, p-chlorobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,p-methoxybenzyloxycarbonyl, benzhydryloxycarbonyl,2-(p-biphenylyl)isopropyloxycarbonyl, 2-(3,5-dimethoxyphenyl)isopropyloxycarbonyl, p-phenylazobenzyloxycarbonyl),triphenylphosphonoethyloxycarbonyl and 9-fluorenylmethyloxycarbonylgroups,

substituted or unsubstituted groups of the alkyloxycarbonyl type such asespecially tert-butyloxycarbonyl, tert-amyloxycarbonyl,diisopropylmethyloxycarbonyl, isopropyloxycarbonyl, ethyloxycarbonyl,allyloxycarbonyl, 2-methylsulphonylethyloxycarbonyl and2,2,2-trichloroethyloxycarbonyl groups,

groups of the cycloalkyloxycarbonyl type such as especiallycyclopentyloxycarbonyl, cyclohexyloxycarbonyl, adamantyloxycarbonyl andisobornyloxycarbonyl groups,

groups derived from heteroatoms, such as benzenesulphonyl,p-toluenesulphonyl (tosyl), mesitylenesulphonyl,methoxytrimethylphenylsulphonyl, o-nitrophenylsulphenyl andtrimethylsilyl groups,

b) as groups protecting the carboxyl functional group, groups such asespecially methyl, ethyl, t-butyl, cyclohexyl, benzyl, p-nitrobenzyl,phenacyl, trimethylsilyl, carboxamidomethyl and fluorenylmethyl groups,

c) as groups protecting the hydroxyl functional group, groups such asespecially t-butyl, tetrahydropyranyl, benzyl, trimethylsilyl,methoxymethyl, trityl and fluorenylmethyl groups,

d) as activating groups especially O-succinimidoyl,O-norbornenedicarboximidoyl, O-benzotriazolyl,O-oxodihydrobenzotriazolyl, O-hydroxypyridyl, pentachlorophenyl,trichlorophenyl, pentafluorophenyl, isobutyloxycarbonyl andtrimethylacetyl groups.

In a first embodiment of the method for producing an N-carbamoyl-peptideaccording to the invention, the intermediate peptide containing one ormore N-ArOC groups is synthesised starting with the N-ArOC derivative ofan amino acid, that is to say by incorporation into the peptide chain ofthe amino acid of which it is desired to convert an amino functionalgroup to a ureido or ureino functional group, directly in the form ofits N-ArOC derivative. Indeed, it emerged, surprisingly, that theseN-ArOC derivatives of amino acids are not only remarkably stable, butare furthermore excellent at preserving the chirality of the compounds,which allows their use in conventional chemical methods for peptidesynthesis both in the solid phase and in the liquid phase. The use, asreagent for synthesising the intermediate peptide, of the amino acidwhich is N-substituted by the ArOC group is particularly advantageous.Indeed, the ArOC group acting, in a remarkably effective manner, as thegroup protecting the amino functional group in question, and also as anintermediate, particularly one which is specific for the desired ureidoor ureino functional group while preserving the chirality of thecompounds, this embodiment of the method according to the inventionmakes it possible to prepare the desired N-carbamoyl-peptide with ayield never achieved before.

The N-ArOC derivative of the amino acid, for which it is desired toproduce in fine the N-carbamoyl derivative inside a peptide, may beconventionally prepared by a method similar to those used for attachingonto an amino functional group a protecting group of thealkyloxycarbonyl or aralkyloxycarbonyl type. A conventional method,known as the Schotten-Baumann procedure, consists in reacting, in anaqueous medium, the sodium form of the amino acid with the desired arylchloroformate. Another procedure which gives excellent results and whichis therefore preferred, consists in using the amino acid in the form ofits persilylated derivative. This persilylated derivative may beproduced for example by a treatment under reflux with an excess amountof trimethylcyanosilane until a homogeneous solution is produced. Thissolution is then diluted, for example with methylene chloride, andcooled to a temperature of less than -10° C., preferably less than orequal to -15° C. The stoichiometric quantity of aryl chloroformate isthen added very slowly, then after reacting for a few minutes, thesolution is concentrated and the N-ArOC-amino acid is isolated via themost appropriate route, depending on its physicochemical properties.

This first embodiment of the method according to the invention ispreferred when it is desired to incorporate in fine, inside a peptide,the N.sup.ω -carbamoyl derivative of a diamino acid. In this specialcase of (α,ω-diamino acids, where it is necessary selectively to blockthe amino functional group of the side chain, the N.sup.ω -ArOCderivative may be prepared by using the well known techniques ofselective acylation, for example by means of the copper(II) complexaccording to a procedure similar to that described especially in"Methoden Der Organischen Chemie" (HOUBEN-WEYL), 1974, Volume XV/1, p.472, with respect to N.sup.ε -benzyloxycarbonyl-L-lysine.

In this specific case of α,ω-diamino acids, depending on the method ofpeptide synthesis and depending on the strategy used, the α-NH₂functional group may be blocked, if necessary, by any customaryprotecting group or may constitute the amine-containing component to beintroduced into a peptide chain whose carboxyl terminal functional groupis activated in a conventional manner. The N.sup.ω -ArOC-amino acid maybe coupled to this chain, for example in silylated form, it beingpossible to carry out the silylation by means especially oftrimethylsilyl chloride and triethylamine, hexamethyldisilazane,bistrimethylsilylacetamide, or trimethylcyanosilane (TMSCN), asdescribed in European Patent EP-B-184243. Any other coupling method mayalso be used such as, for example, the use of the N.sup.ω -ArOC-aminoacid as it is, in combination with an active ester, in a semi-aqueousmedium.

Similarly, depending on the method of peptide synthesis and depending onthe strategy used, the carboxyl functional group of any N-ArOC-aminoacid may be optionally blocked by a customary protecting group oractivated by a conventional activating agent.

After incorporation of the N-ArOC-amino acid into a peptide fragment,the peptide synthesis may be continued in a conventional manner bycondensation between a fragment reacting via its amino functional group(amine-containing component) and a fragment reacting via its carboxylfunctional group (carboxylic component). The presence of the N-ArOCgroup, both on the amine-containing component and on the carboxyliccomponent does not in any way interfere either with the coupling withother amino acids or peptide fragments, or with the methods ofactivation normally used.

This first embodiment of the method according to the invention isparticularly preferred for synthesising peptides containing L, D, DL2-amino-4-ureido-butyric (norcitrulline), citrulline and homocitrullineresidues from the corresponding L, D or DL precursor amino acidsrespectively, that is to say from 2,4-diaminobutyric acid, ornithine andlysine respectively. These amino acids may be introduced withoutdifficulty into an intermediate peptide chain, preferably directly inthe form of their N.sup.ω -PhOC or N.sup.ω -Toc derivatives, whichderivatives are subsequently converted in situ inside the peptide or apeptide fragment to an N.sup.ω -carbamoyl derivative with a very highyield and, in the case of the D or L structures, without modification ofthe chiral purity, which is a key element in physiological ortherapeutic activity.

In a second embodiment of the method for producing N-carbamoyl-peptidesaccording to the invention, the intermediate peptide containing one ormore N-ArOC groups is synthesised starting with a precursor peptide, byintroducing the ArOC group onto the amino functional group(s) to becarbamoylated inside the peptide chain and not in the amino acid. Thisembodiment of the method may be used especially to carbamoylate one ormore free amino functional groups of a natural peptide, but may also beused in the case of synthetic peptides. In this latter case, theintermediate peptide is prepared in two successive stages: a first stageconsists in the synthesis of the precursor peptide containing the aminoacid residue bearing the amino functional group to be carbamoylated,starting with the various amino acids forming it, and a second stageconsisting in the conversion of the precursor peptide to an intermediatepeptide containing an N-ArOC group. The amino acid of which it isdesired to convert an amino functional group to a ureido or ureinofunctional group is incorporated into the peptide chain of the precursorpeptide in a conventional manner, masking this amino functional groupduring the peptide synthesis by a customary protecting group, forexample by a benzyloxycarbonyl, tert-butyloxycarbonyl or phthaloylgroup. At a subsequent stage of the synthesis, after incorporation intothe peptide chain of the amino acid residue containing the protectedamino functional group and at the most appropriate time depending on thestrategy adopted, the customary group protecting this functional groupis removed in the conventional manner so as to obtain the precursorpeptide.

In the natural or synthetic precursor peptide, the amino functionalgroup which it is desired to carbamoylate, at that time in the freeform, is then converted, in a second stage, to an ArOC-amino functionalgroup to form the intermediate peptide. The introduction of this ArOCgroup is carried out by reaction of this free amino functional groupwith an aryloxycarbonylating agent, for example with an arylchloroformate or with an aryloxycarbonyloxysuccinimide. In the casewhere the amino functional group to be carbamoylated is a ω-aminofunctional group, the introduction of the ArOC group may beconventionally carried out by a method similar to those used forattaching onto an amino functional group a conventional protectinggroup, such as the benzyloxycarbonyl group. In the case where the aminofunctional group to be carbamoylated is a α-amino functional group, oneprocedure for introducing the N-ArOC group which gives excellent resultsand which is therefore preferred, consists in using the precursorpeptide in the form of its persilylated derivative, which derivative maybe obtained in a manner known per se, by reaction with a silylatingagent considered above. The persilylated derivative is placed in contactwith the aryloxycarbonylating agent, preferably in stoichiometricproportions, in a water-immiscible solvent. Suitable solvents areespecially methyl tert-butyl ether, dichloromethane and ethyl acetate.This latter solvent is preferred. The reaction between the persilylatedderivative and the aryloxycarbonylating agent is carried out at atemperature of 0° to 40° C., preferably at room temperature. Afterreacting for a few minutes, preferably not more than two minutes, wateris added to the reaction medium and the two-phase system obtained isstirred for a period of about 10 to 30 minutes. After separating thephases, the intermediate peptide containing the N-ArOC group is thenisolated from the organic phase by the most appropriate route, accordingto its physicochemical properties. This preferred procedure makes itpossible to prepare the intermediate peptide in a very selective manner,while preserving the chirality of the compounds and avoiding theformation of by-products of a heterocyclic nature.

Preferably, after purification of the intermediate peptide containingthe N-ArOC group(s) by any known method, for example by crystallisation,the said peptide is placed in contact with a compound R1R2NH as definedabove, to give, by the method according to the invention, the peptide inwhich the free amino functional group of the precursor peptide has beenconverted to a ureino functional group.

Using a conventional group for protecting the amino functional group tobe carbamoylated in order to introduce this amino acid into the peptidechain of the precursor peptide, which protecting group must subsequentlybe removed and replaced by an ArOC group to give the intermediatepeptide, this second embodiment of the method according to the inventioninvolves, for a synthetic peptide, a greater number of steps than thefirst embodiment of the method in which the intermediate N-ArOC-peptideis prepared from the outset with an N-ArOC amino acid. Consequently,when the peptide to be carbamoylated is a peptide prepared by thesynthetic route starting with the constituent amino acids, the firstembodiment of the method for producing N-carbamoyl-peptides according tothe invention is preferred. On the other hand, in the case of naturalpeptides, the second embodiment of the method for producingN-carbamoyl-peptides according to the invention is preferred.

Regardless of the method for incorporating the N-ArOC group into thepeptide structure, the amino acid residue bearing an N-ArOC group isthen converted in situ, inside the peptide chain, at a time judged mostappropriate, by reaction with a compound R1R2NH as defined above, togive, by the method according to the invention, the correspondingN-carbamoyl-peptide.

The compound R1R2NH is used in excess relative to the intermediateN-ArOC-peptide, generally in a quantity 2 times greater than thestoichiometric quantity, the most often in a quantity 5 to 100 times,preferably 5 to 20 times, greater than the stoichiometric quantity. Thereaction is carried out in any appropriate medium, especially in anorganic solvent such as tetrahydrofuran, dimethylformamide, ethylacetate or an alcohol such as methanol. The compound R1R2NH is usedeither in anhydrous form, or in concentrated aqueous solution. Thereaction is generally carried out at a temperature of -20° to +50° C.Preferably, the method according to the invention is carried out at atemperature of 0° to 20° C., such a temperature making it possible toobtain a good reaction rate while preserving a very high selectivity.

It is evident that the other protecting groups which may be present inthe intermediate peptide structure during the use of the methodaccording to the invention must be stable under the conditions ofammonolysis or of aminolysis of the N-ArOC group, unless it is desiredto take advantage of the ammonolysis or the aminolysis of the methodaccording to the invention to remove at the same time the otherprotecting groups still present in the peptide structure.

The invention also relates to the diamino acids N.sup.ω -substituted byan ArOC group and the intermediate peptides containing one or morediamino acid residues N.sup.ω -substituted by an ArOC group. Thesediamino acids and intermediate peptides may be optionally substituted,protected or activated. Consequently, the invention relates to thecompounds corresponding to the general formula ##STR2## in which R3represents an aryl group which is unsubstituted or substituted by one ormore alkyl groups containing 1 to 4 carbon atoms

R4 represents a hydrogen atom, a group for protecting the aminofunctional group, an amino acid or a peptide some of whose functionalgroups are optionally substituted by a protecting group or by anactivating group

R5 represents a hydroxyl group, a halogen atom, a group for protectingthe carboxyl functional group, an activating group, an amino group, anamino acid or a peptide some of whose functional groups are optionallysubstituted by a protecting group or by an activating group

n is an integer from 1 to 10.

As nonlimitative examples of groups for protecting the amino functionalgroup which may be represented by R4, there may be mentioned especiallysubstituted or unsubstituted groups of the alkyl or aralkyl type, suchas benzyl, diphenylmethyl, di(methoxyphenyl)methyl and triphenylmethyl(trityl) groups, substituted or unsubstituted groups of the acyl typesuch as formyl, acetyl, trifluoroacetyl, benzoyl and phthaloyl groups,substituted or unsubstituted groups of the aralkyloxycarbonyl type suchas benzyloxycarbonyl, p-chlorobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,p-methoxybenzyloxycarbonyl, benzhydryloxycarbonyl,2-(p-biphenylyl)isopropyloxycarbonyl,2-(3,5-dimethoxyphenyl)isopropyloxycarbonyl,p-phenylazobenzyloxycarbonyl), triphenylphosphonoethyloxycarbonyl and9-fluorenylmethyloxycarbonyl groups, substituted or unsubstituted groupsof the alkyloxycarbonyl type such as tert-butyloxycarbonyl,tert-amyloxycarbonyl, diisopropylmethyloxycarbonyl,isopropyloxycarbonyl, ethyloxycarbonyl, allyloxycarbonyl,2-methylsulphonylethyloxycarbonyl and 2,2,2-trichloroethyloxycarbonylgroups, groups of the cycloalkyloxycarbonyl type such ascyclopentyloxycarbonyl, cyclohexyloxycarbonyl, adamantyloxycarbonyl andisobornyloxycarbonyl groups, groups containing a heteroatom, such asbenzenesulphonyl, p-toluenesulphonyl (tosyl), mesitylenesulphonyl,methoxytrimethylphenylsulphonyl, o-nitrophenylsulphenyl andtrimethylsilane groups.

As nonlimitative examples of groups for protecting the carboxylfunctional group which may be represented by R5, there may be mentionedespecially methyl, ethyl, t-butyl, cyclohexyl, benzyl, p-nitrobenzyl,phenacyl, trimethylsilyl, carboamidomethyl and fluorenylmethyl groups.

As nonlimitative examples of activating groups which may be representedby R5, there may be mentioned especially O-succinimidoyl,O-norbornenedicarboximidoyl, O-benzotriazolyl,O-oxodihydrobenzotriazolyl, O-hydroxypyridyl, pentachlorophenyl,trichlorophenyl, pentafluorophenyl, isobutyloxycarbonyl andtrimethylacetyl groups.

Generally, R3 is a group containing at most 12 carbon atoms. Most often,R3 is a phenyl, naphthyl, tolyl, xylyl, mesitylyl, ethylphenyl,diethylphenyl, propylphenyl or isopropylphenyl group. Preferably, R3 isa phenyl or tolyl group. In a particularly preferred manner, R3 is aphenyl or p-tolyl group.

Preferably, n is an integer from 2 to 6. In a particularly preferredmanner, n is an integer from 2 to 4.

Finally, compounds which give very good results in the method accordingto the invention are especially those where:

R3 represents a phenyl group

R4 represents a hydrogen atom or the dipeptide Z-Ser-Tyr-

R5 represents a hydroxyl group, an amino acid or a peptide some of whosefunctional groups are optionally substituted by a protecting group or byan activating group

n is equal to 2, 3 or 4.

These preferred compounds according to the invention are N.sup.ω -PhOCderivatives of 2,4-diaminobutyric acid, ornithine or lysine and peptidesor peptide structures containing an N.sup.ω -PhOC derivative of aresidue of one of these diamino acids, regardless of their configuration(D, L or DL).

As examples, there may be mentioned, as compounds according to theinvention, N.sup.γ -PhOC-diaminobutyric acid and the peptides containinga diaminobutyric residue in any position, whose γ-amino functional groupis substituted by a PhOC group; N.sup.δ -PhOC-D-ornithine and thepeptides containing a D-ornithine residue in any position, whose δ-aminofunctional group is substituted by a PhOC group; N.sup.ε -PhOC-lysineand the peptides containing a lysine residue in any position, whoseε-amino functional group is substituted by a PhOC group. A peptidecontaining a diamino acid residue N.sup.ω -substituted by an ArOC groupis for example the tripeptide Z-Ser-Tyr-D-Orn(PhOC).

These compounds according to the invention are excellent synthesisintermediates for the preparation of various peptides including aminoacid residues bearing a ureino group, such as the citrulline,homocitrulline, 2-amino-4-ureidobutyric residues: the N.sup.ω -ArOCamino acids may be easily incorporated into intermediate peptidestructures, which structures may then be easily converted to N.sup.ω-carbamoyl-peptides by the method according to the invention.

In particular, these compounds are excellent intermediates for themanufacture, in liquid phase, of fragments of modified hormones.

The invention also relates to the peptides of general formula

    R6-Ser(R7)-Tyr(R8)-R9-R10

in which

R6 is a hydrogen atom or a group for protecting the amino functionalgroup,

Ser(R7) is a serine residue with R7 representing a hydrogen atom or agroup for protecting the hydroxyl functional group of the side chain ofthe serine residue

Tyr(R8) is a tyrosine residue with R8 representing a hydrogen atom or agroup for protecting the hydroxyl functional group of the side chain ofthe tyrosine residue

R9 is, in a first variant, a 2,4-diaminobutyric or ornithine residue ofthe D, L or DL configuration, and in a second variant, a2-amino-4-ureidobutyric, citrulline or homocitrulline residue of the D,L or DL configuration, in which the hydrogen atoms of the carbamoylfunctional group are optionally substituted by an alkyl, cycloalkyl oraralkyl group containing at most 12 carbon atoms, or by a cycloalkylenegroup containing 3 to 6 carbon atoms.

R10 is a hydroxyl radical, an amino acid or a peptide some of whosefunctional groups are optionally substituted by a protecting group.

As nonlimitative examples of groups for protecting the amino functionalgroup, which may be represented by R6, there may be mentioned especiallybenzyloxycarbonyl and tert-butyloxycarbonyl groups.

As nonlimitative examples of groups for protecting the hydroxylfunctional group of the side chain of the serine residue or the tyrosineresidue, which may be represented by R7 or by R8, there may be mentionedespecially benzyl, benzyloxycarbonyl, trimethylsilyl groups.

Preferably, R9 represents residues with a D configuration. Preferably,R10 is a hydroxyl radical or a peptide fragment containing 1 to 5 aminoacids. In a particularly preferred manner, R10 is a hydroxyl radical.

When R9 is an amino acid residue according to the first variant, thepeptides are especially precursors of some intermediate peptidescontaining an N-aryloxycarbonyl group used in the method according tothe invention. The preparation of these precursor peptides may becarried out by any conventional method of peptide synthesis, either in"solid" phase according to the technique described by Merrifield, or inliquid phase.

When R9 is an amino acid residue according to the second variant, thepeptides are especially fragments of some modified hormones. Thepreparation of these peptide fragments may be easily carried out inliquid phase by means of the method according to the invention, by usingtheir N.sup.ω -ArOC intermediates according to the invention, optionallyobtained starting with the precursor peptides above.

The symbolic representations of the amino acids and peptides adopted inthe description and the examples follow the IUPAC recommendations onnomenclature, which are generally adopted and described for example in"Nomenclature and Symbolism for Amino Acids and Peptides,Recommendations 1983", Eur. J. Biochem. (1984), 138, p. 9-37. Byconvention, the peptides are represented with their N-terminal end tothe left and their C-terminal end to the right. The abbreviations usedto designate the amino acid residues are derived from the trivial namesof the amino acids and are A₂ bu, 2,4-diaminobutyric acid; Orn,ornithine; Lys, lysine; Nci, 2-amino-4-ureidobutyric acid ornorcitrulline; Cit, citrulline; Hci, homocitrulline; Ser, serine; Tyr,tyrosine; Leu, leucine; Arg, arginine; Pro, proline; Gly, glycine; Ala,alanine. Unless otherwise stated, all the amino acids described areamino acids of the L form.

The various products and synthesis intermediates reported in theexamples were characterised by various analytical methods which are usedunder the following conditions:

thin-layer chromatography (TLC):

silica gel plates MERCK 60F-254

eluents:

Rf(1) HCOOMe:HCOOH:MeOH 95:2.5:2.5

Rf(2) EtOAc:EtOH:HOAc 8:1:1

Rf (3) CH₃ CN:CHCl₃ :HOAc;H₂ O 7:7:4:2

Rf(4) HCOOMe;HCOOH:MeOH:H₂ O 92.5:2.5:2.5:2.5

HPLC chromatography:

column: Vydac 5μ C18

elution: gradient from 98% A+2% B up to 25% A+75% B in 49 minutes (A=H₂O containing 0.1% TFA; B=CH₃ CN containing 0.1% TFA) .

flow rate=2 ml/min

detection: UV 220 nm.

nuclear magnetic resonance (NMR):

apparatus: Bruker AMX 500 MHz

shift given in ppm

course of the resonances: m=multiplet, s=singlet, d=doublet, t=triplet,q=quadruplet, quint=quintuplet.

The following abbreviations are used in the examples below:

α=optical rotation, measured at 589 nm at 25° C.

CH₃ CN=acetonitrile

DMF=dimethylformamide

EtOAc=ethyl acetate

EtOH=ethyl alcohol

HCOOH=formic acid

HCOOMe=methyl formate

HOAc=acetic acid

i.BuOCOCl=isobutyl chloroformate

MeOH=methyl alcohol

MTBE=methyl tert-butyl ether

NMM=N-methylmorpholine

NMP=N-methylpyrrolidone

PhEt=phenylethyl

PhOC=phenyloxycarbonyl

PhOC-OSu=phenyloxycarbonyloxysuccinimide

Pht=phthaloyl

PivCl=pivaloyl chloride

m.p.=melting point

Pyr=pyridine

TEA=triethylamine

TFA=trifluoroacetic acid

THF=tetrahydrofuran

TMS=trimethylsilyl

TMSCN=trimethylcyanosilane

Z=benzyloxycarbonyl

DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLE 1

Preparation of N.sup.ε -PhOC-lysine from lysine

A solution containing 36.5 g of LysHCl and 16 g of NaOH in 200 ml ofwater is mixed with 25 g of CuSO₄.5H₂ O dissolved in 80 ml of water. 25g of NaHCO₃ are then introduced, with stirring.

After dissolution of the bicarbonate, the solution is cooled to 0° C.and then treated dropwise with 30 ml of phenyl chloroformate (durationof the introduction 1 h). After allowing to stand for 3 h at roomtemperature, the precipitate formed is filtered, washed twice with 200ml of water, twice with 200 ml of acetone and then air dried. Thecopper(II) complex thus obtained is suspended in 400 ml of water andthen decomposed by the addition of 80 ml of concentrated HCl. Thecopper(II) ions are then precipitated by the addition, in smallportions, of the stoichiometric quantity of sodium sulphide. Afterdegassing in order to remove the H₂ S which may have formed, thesuspension is mixed with 10 g of celite.

After filtration and washing of the cake with 200 ml of water, the pH ofthe filtrate is adjusted to 7 by introduction of NaHCO₃. N.sup.ε-PhOC-Lys precipitates. After 1 h at 0° C., it is collected byfiltration, washed with water, with methanol and with acetone and thendried. Yield: 45 g (84%)

m.p.: 211°-215° C. (decomp.

α:+16.6 (c=1/1N HCl)

TLC: Rf (3)=0.50

HPLC: t_(R) =11.70 min

NMR (¹ H): (ref. DMSO-d₆ at 2.49 ppm; product dissolved in DMSO-d₆ byadding a drop of TFA)

8.27 (3H broad s) NH3 7.70 (1H broad s) NH

7.35 (2H t) m-phenyl 7.18 (1H t) p-phenyl

7.08 (2H d) o-phenyl 3.89 (1H broad s) Hα

3.06 (2H m) Hε

1.79 (2H m), 1.48 (3H m) and 1.37 (1H m) Hβ, Hγ and Hδ.

EXAMPLE 2

Preparation of N.sup.δ -PhOC-D-ornithine from D-ornithine

N.sup.δ -PhOC-D-ornithine was prepared, with an identical yield,according to the same procedure as N.sup.ω -PhOC-lysine of Example 1.

m.p.=197°-199° C. (decomp.)

α=-17.8 (c=1.1/1N HCl)

TLC: Rf (3)=0.54

HPLC: t_(R) =9.69 min.

EXAMPLE 3

Preparation of N.sup.γ -PhOC-D-diaminobutyric acid from D-diaminobutyricacid

N.sup.γ -PhOC-D-diaminobutyric acid was prepared, with a yield of 65%according to the same procedure as N.sup.ε -PhOC-lysine of Example 1.

m.p.=202°-204° C.

TLC: Rf (3)=0.60

HPLC: t_(R) =7.55 min.

EXAMPLE 4

Synthesis of Z-Ser-Tyr-D-Orn(PhOC)

4.03 g (10 mmol) of Z-Ser-Tyr are dissolved in 15 ml of THF and thenneutralised with 1.10 ml (10 mmol) of NMM. The activation of the peptideis carried out at -15° C. by addition of 1.3 ml (10 mmol) of i.BuOCOCl.After 3 minutes, the coupling is carried out by addition of a solution,cooled to 0° C., of persilylated N.sup.ε -PhOC-D-ornithine, preparedfrom a suspension of 3.15 g (12.5 mmol) of N.sup.δ -PhOC-D-ornithine in6.5 ml (50 mmol) of TMSCN which is refluxed until a clear solution isobtained.

After reacting for 5 minutes at low temperature, the temperature isincreased to 20° C. and the mixture is allowed to stand for 1/2 h. Thereaction is stopped by addition of 60 ml of EtOAc and 50 ml of a 5%aqueous solution of KHSO₄. After removing the aqueous phase, the organicphase is washed with 20 ml of water and then concentrated to dryness.The residue is dissolved in 60 ml of methanol and the crystallisation ofthe tri-peptide, in the form of an ammonium salt, is induced by additionof concentrated ammonium hydroxide up to pH 8.

Yield: 5.1 g (78%)

Analytical data for a sample released from its salt:

m.p.: 140°-160° C. (decomp.)

α: -23.3 (c=1.06/MeOH)

TLC: Rf (1)=0.76/Rf (2): 0.70

HPLC: t_(R) =23.43 min

NMR (¹ H) (ref. CD₃ OD at 3.31):

    ______________________________________                                        7.30 (7 H m) 5 H of Z, 2 H of PhOC                                                                 7.19 (1 H t) p-PhOC                                      7.08 (4 H m) 2 H of PhOC, 2 H of Tyr                                                               6.71 (2 H d) 2 H of Tyr                                  5.10 (2 H q) phenylCH.sub.2 O                                                                      4.67 (1 H q) TyrHα                                 4.41 (1 H q) OrnHα                                                                           4.17 (1 H t) SerHα                                 3.79 (2 H m) Serβ's                                                                           3.16 (2 H t) Ornδ's                                3.10 (1 H q) TyrβA                                                                            2.90 (1 H q) TyrβB                                  1.89 (1 H m) 1.74 (1 H m)                                                     1.50 (2 H m) OrnHβ's + OrnHγ's.                                    ______________________________________                                    

EXAMPLE 5

Synthesis of Z-Ser-Tyr-D-Cit

2.0 g (3.15 mmol) of Z-Ser-Tyr-D-Orn(PhOC) obtained in Example 4 areadded to a mixture of 20 ml of MeOH and 20 ml of 25% NH₄ OH. Thesolution obtained is kept at 40° C. for 75 minutes and then evaporatedto dryness. The residue is resuspended in a mixture of 10 ml of waterand 10 ml of EtOAc. The crystallisation of the tripeptide Z-Ser-Tyr-Citis induced by acidifying the aqueous phase (pH 3) by means of aconcentrated solution of KHSO₄. The peptide is collected by filtrationand then dried. Yield: 1.45 g (82%). The analytical data for thetripeptide Z-Ser-Tyr-D-Cit produced are:

m.p.: 132°-138° C. (decomp.)

α: -8.10 (c=1.1/DMF)

TLC: Rf (4)=0.17

HPLC: t_(R) =16.44 min

NMR (¹ H):

Spectrum identical to that for Z-Ser-Tyr-D-Orn-(PhOC) (Example 4), butdisappearance of the resonances of the PhOC group and appearance of theresonances of the carbamoyl group:

5.90 (1H t) NH 5.36 (2H s) NH₂

EXAMPLE 6

Synthesis of Z-Ser-Tyr-D-Cit(PhEt)

654 mg (1 mmol) of Z-Ser-Tyr-D-Orn(PhOC), crystallised in the form ofits ammonium salt, is suspended in a two-phase system composed of 10 mlof 2% KHSO₄ and 20 ml of EtOAc. The mixture is stirred at 50° C. for 1hour. The organic phase is recovered, concentrated to one half, thentreated with 1.22 g (10 mmol) of phenylethylamine. The progress of thereaction can be monitored by TLC. As soon as conversion of thetripeptide is complete, the reaction medium is concentrated to drynessand the solid obtained washed twice with 20 ml of MTBE. The tripeptideis thus recovered in the form of the phenylethylamine salt(Z-Ser-Tyr-D-Cit(PhEt)-NHPhEt). In order to obtain the free tripeptide,the salt it dissolved in a minimum amount of methanol and thenprecipitated in a large volume of a 5% solution of KHSO₄. Afterfiltration and washing with water, the tripeptide Z-Ser-Tyr-D-Cit(PhEt)is obtained.

Yield: 630 mg (95%)

m.p.: 160°-169° C. (decomp.)

α: -21.7 (c=1/MeOH)

TLC: Rf (1)=0.65/Rf (2)=0.51

HPLC: t_(R) =23.59 min

NMR (¹ H) (ref. DMSO-d₆ at 2.49 ppm):

9.12 (1H s) OHTyr 8.16 (1H d) NHOrn 7.91 (1h d) NHTyr

7.35 (5H m) H's Z 7.28 (2H m) +7.17 (3H m) H's of Ph.ethylamine

7.21 (1H d) NHSer 6.99 (2H d) Hδ'sTyr 6.60 (2H d) Hε'sTyr

5.84 (1H t) NH ureido-Orn

5.77 (1H t) NH ureido-phenylethylamine

5.01 (2H q) CH₂ Z 4.48 (1H m) HαTyr 4.13 (1H m) HαOrn

4.05 (1H m) HαSer 3.48 (2H d) Hβ'sSer

3.19 (2H q) NHCH₂ phenylethylamine 2.92 (2H q) Hδ'sOrn

2.87 (1H q) HβATyr 2.69 (1H q) HβBTyr

2.64 (2H t) CH₂ Ph. phenylethylamine 1.63 (1H m) HβAOrn

1.50 (1H m) HβBOrn 1.28 (2H m) Hγ'sOrn

EXAMPLE 7

Synthesis of Z-Ser-Tyr-D-Cit(2-methylbutyl)

The synthesis of Example 6 was repeated using 2-methylbutylamine inplace of phenylethylamine. Z-Ser-Tyr-D-Cit(2-methylbutyl) was obtained.

TLC: Rf (1)=0.63/Rf (2)=0.52

HPLC: t_(R) =23.07 min

NMR (¹ H): compared with the starting material (Example 4), the linesfor the PhOC group disappeared and the lines for the 2-methylbutylamineresidue appeared at 3.03 and 2.90 for the N--CH₂ --; at 1.40 for theN--CH₂ --CH(CH₃)--CH₂ --; at 1.12 for the N--CH₂ --CH--, at 0.89 for theN--CH₂ --CH(CH₃)--CH₂ --CH₃ and at 0.87 for the N--CH₂ --CH(CH₃)--

Example 8

Synthesis of Z-Ser-Tyr-D-Cit(tetramethylene)

The synthesis of Example 6 was repeated using pyrrolidine in place ofphenylethylamine. Z-Ser-Tyr-D-Cit(tetramethylene) was obtained.

TLC: Rf (1)=0.35/Rf (2)=0.21

HPLC: t_(R) =19.18 min

NMR (¹ H): the lines due to the pyrrolidine residue are situated at 3.30(4H) and at 1.87 (4H)

EXAMPLE 9

Synthesis of Z-Ser-Tyr-D-Orn(PhOC) from Z-Ser-Tyr-D-Orn

a) Synthesis of Z-Ser-Tyr-D-Orn(Pht)

The tripeptide Z-Ser-Tyr-D-Orn(Pht) is first prepared by reaction ofZ-Ser-Tyr with δ-Pht-D-ornithine according to a procedure similar tothat of Example 4. The desired tripeptide is obtained in the form of anammonium salt, with a yield of 85%.

Analytical data:

HPLC: t_(R) =24.06 min

NMR (¹ H) (in DMSO-d₆):

    ______________________________________                                        7.80 (4 H, m) H'sPht                                                                      7.31 (5 H, m) H's Z                                                                         6.96 (2 H, d) HδTyr                           6.60 (2 H, d) HεTyr                                                               4.96 (2 H, q) CH.sub.2 -Z                                                                   4.41 (1 H m) HαTyr                            4.15 (1 H, m) HαOrn                                                                 3.99 (1 H, m) HαSer                                                                   3.53 (2 H, m) HδOrn                           3.44 (2 H, d) HβSer                                                                  2.83 (1 H dd) HβATyr                                                                   2.67 (1 H dd) HβBTyr                           1.65 (1 H m) +                                                                1.52 (3 H m) Hβ +                                                        HγOrn                                                                   ______________________________________                                    

b) Preparation of Z-Ser-Tyr-D-Orn

The side group of the ornithine residue is deprotected byhydrazinolysis: 28 g of Z-Ser-Tyr-D-Orn(Pht) are suspended in 1.3 L ofMeOH. After addition of 10 ml of NH₂ NH₂.H₂ O, the mixture is heated toboiling temperature. After 2 hours, the reaction product begins tocrystallise. One hour later, the heating is stopped and the mixture iscooled to room temperature. The precipitate is collected by filtration.After washing with water and with cold methanol, the cake is dried so asto give 90% of pure Z-Ser-Tyr-D-Orn.

HPLC : t_(R) =15.37 min

NMR(¹ H) (in CD₃ OD):

7.34 (5H m) H-arom. Z 7.08 (2H d) HδTyr 6.73 (2H m) HεTyr

5.09 (1H d) and 4.98 (1H d) CH₂ --Z 4.59 (1H m) HαTyr

4.45 (1H m) HαOrn 4.13 (1H m) HαSer 3.68 (2H m) HβSer

3.13 (1H dd) HβATyr 2.90 (3H m) HβBTyr+HδOrn

1.94 (1H m), 1.77 (1H m) and 1.63 (2H m) Hβ+HγOrn

c) Synthesis of Z-Ser-Tyr-D-Orn(PhOC)

The PhOC group is introduced into the side chain of the ornithineresidue in the following manner:

5.17 g (10 mmol) of Z-Ser-Tyr-D-Orn are dissolved in 50 ml of a 1:1THF:H₂ O mixture by volume. Then the pH is adjusted to 7.5 by additionof TEA. 3.54 g (15 mmol) of PhOC-OSu are then introduced into thereactor. The conversion is complete after allowing the mixture to standfor a period of 2 h 30 min. The THF is removed under vacuum, then 100 mlof EtOAc and, with stirring, 25 ml of a 10% aqueous solution of KHSO₄are added. The decanted organic phase is washed with 50 ml of water, andthen concentrated to dryness. The residue is taken up in 50 ml of MeOHand the pH is adjusted to 8 by means of concentrated ammonium hydroxide.The tripeptide Z-Ser-Tyr-D-Orn(PhOC) crystallises in the form of anammonium salt. It is identical to the produce synthesised via the directroute described in Example 4.

EXAMPLE 10

Synthesis of NH₂ -CO-Ala-Phe

a) Synthesis of PhOC-Ala

20 mmol of alanine are treated under reflux with 30 mmol of TMSCN untilcomplete solubilisation is obtained. After dilution with 30 ml ofdichloromethane, the mixture is cooled to -15° C. 20 mmol of phenylchloroformate are then slowly added. After 10 minutes, the solution isconcentrated to dryness, the residue is taken up in 40 ml ofdichloromethane and washed with 50 ml of a 5% aqueous solution of citricacid and then with 50 ml of water. The organic phase is concentrated,taken up in 40 ml of hot sulphuric ether, and then diluted with hexaneuntil the mixture becomes torbid. After overnight storage in arefrigerator, the crystals formed are filtered, washed and dried to give3.6 g of PhOC-Ala (yield=86%).

m.p.: 119°-124° C.

α: -45.6 (c=1/MeOH)

HPLC: t_(R) =13.45 min

NMR(¹ H): (In CDCl₃ ; for some protons, two forms appear, Major=Mminor=m):

    ______________________________________                                        7.36 (2 H t) PhOC meta 7.21 (1 H t) PhOC                                                           7.14 (2 H d) PhOc ortho                                  para                                                                          6.51 (d) NH-m and 5.62 (d) NH-M                                                                    4.49 (1 H quint) Hα                                1.60 (d) CH.sub.3 -m 1.55 (d) CH.sub.3 -M                                     ______________________________________                                    

b) Synthesis of PhOC-Ala-Phe

2.1 g (10 mmol) of PhOC-Ala, dissolved in 10 ml of dichloromethane areneutralised with 1.1 ml (10 mmol) of NMM. The solution, cooled to -10°C., is treated with 1.3 ml (10 mmol) of i.BuOCOCl. After activating for4 minutes at -10° C., the solution of persilylated phenylalanine isadded and after allowing to stand for 1 hour at room temperature, thedipeptide formed is desilylated by addition of 0.2 ml of water. Then themixture is washed with 100 ml of a 3% aqueous solution of KHSO₄. ThePhOC-Ala-Phe crystallises from the two-phase system. After overnightstorage in a refrigerator, it is recovered by filtration. Yield: 3.1 g(86%)

m.p.: 143°-144° C.

α: -19.5 (c=1/MeOH)

HPLC: t_(R) =22.63 min

NMR (¹ H) (in CDCl₃)

    ______________________________________                                        7.3 to 7.1 (10 H m)       6.74 (1 H d) NH Phe                                 H aromat. PhOC +                                                              Phe                                                                           6.12 (1 H d) NH Ala                                                                       4.77 (1 H q) HotPhe                                                                         4.23 (1 H quint) HαAla                        3.20 (1 H dd) HβAPhe                                                                 3.06 (1 H dd) HβBPhe                                         ______________________________________                                    

c) conversion to NH₂ --CO-Ala-Phe

30 mg of PhOC-Ala-Phe are dissolved in 0.5 ml of a 1:1 mixture by volumeof concentrated ammonium hydroxide (25%) and MeOH. After 3 hours, HPLCanalysis indicates a complete and selective conversion (by-products<2%). The reaction medium is diluted with THF until the mixture becomesturbid. After overnight storage in a refrigerator, a theoreticalquantity of NH₂ -CO-Ala-Phe is recovered in the form of an ammonium saltby filtration.

α: 33.1 (c=O,4/H₂ O)

HPLC: t_(R) =10.14 min

NMR (¹ H) in DMSO-d₆ :

    ______________________________________                                        7.48 (1 H d) NHPhe                                                                        7.13 (5 H m) H arom.Phe                                                                      6.33 (1 H d) NHAla                                 5,62 (2 H s) NH.sub.2 -CO                                                                 4.08 (1 H q) HαPhe                                                                     3.99 (1 H quint)                                   HαAla                                                                   3.07 (1 H dd) HβAPhe                                                                 2.89 (1 H dd) HβBPhe                                                                    1.10 (3 H d) CH.sub.3 Ala.                         ______________________________________                                    

EXAMPLE 11

Synthesis of PhOC-Phe-Val

To 2.04 g (5 mmol) of persilylated Phe-Val (TMS-Phe-Val-OTMS) is added asolution of 20 ml of EtOAc containing 1.17 g (5 mmol) of PhOC-OSu. After2 minutes, 15 ml of water are added. The two-phase system is stirred for15 minutes and then the aqueous phase is removed. The organic phase isextracted twice with 10 ml of water. The residue of the organic phaseafter evaporation consists of 1.6 g of PhOC-Phe-Val, contaminated withtrace amounts of Phe-Val. By taking up this residue in a two-phasewater/MTBE system, the free dipeptide is extracted into the aqueousphase and the residue of the organic phase is PhOC-Phe-Val with apeptide purity greater than 95%.

TLC: Rf(2)=0.86

HPLC: t_(R) =25.2 min

NMR (¹ H) (ref. CD₃ OD at 3.30 ppm)

    ______________________________________                                        7.30 (7 H m) 5H Phe, 2H m-PhOC                                                                    7.17 (1 H t) p-PhOC                                       6.98 (2 H d) o-PhOC 4.56 (1 H t) Hα Phe                                 4.39 (1 H d) Hα Val                                                                         3.22 (1 H dd) Hβ-1 Phe                               2.92 (1 H dd) Hβ-2 Phe                                                                       2.20 (1 H m) Hβ Val                                  1.00 (6 H m) 2 × CH.sub.3 Val                                           ______________________________________                                    

What is claimed is:
 1. A method for producing a stereochemically preserved peptide containing at least one N-carbamoyl functional group comprising reacting an intermediate peptide containing one or more aryloxycarbonyl groups consisting of a group of general formula ##STR3## attached to the nitrogen atom of an amino functional group and in which R3 is selected from the group consisting of phenyl, naphthyl, tolyl, xylyl, mesitylyl, ethylphenyl, diethylphenyl, propylphenyl and isopropylphenyl with a compound of general formula R1R2NH in which R1 and R2 represent, independently of each other, hydrogen atoms, alkyl, cycloalkyl or aralkyl radicals, containing at most 12 carbon atoms, or in which R1 and R2 together form an alicyclic radical containing 3 to 6 carbon atoms, to produce said peptide.
 2. The method according to claim 1, wherein R3 is a phenyl or p-tolyl group.
 3. The method according to claim 1, wherein the intermediate peptide bears an aryloxycarbonyl group on one or more ω-amino functional groups.
 4. The method according to claim 1, wherein the intermediate peptide is synthesised starting with the N-aryloxycarbonyl derivative of an amino acid.
 5. The method according to claim 4, wherein the intermediate peptide is synthesised starting with the N.sup.ω -aryloxycarbonyl derivative of a diamino acid.
 6. The method according to claim 1, wherein the intermediate peptide is synthesised starting with a precursor peptide, by introducing the aryloxycarbonyl group onto the amino functional group(s) to be carbamoylated inside the peptide chain.
 7. The method according to claim 1, wherein R3 is phenyl.
 8. The method according to claim 1, wherein the compound R1R2NH is used in a quantity 2 to 100 times greater than a stoichiometric quantity.
 9. A method for producing a peptide containing at least one N-carbamoyl functional group, comprising the steps of:a) synthesizing an intermediate peptide containing at least one aryloxycarbonyl group consisting of a group of general formula ##STR4## attached to a nitrogen atom of an amino functional group and in which R3 is selected from the group consisting of phenyl, naphthyl, tolyl, xylyl, mesitylyl, ethylphenyl, diethylphenyl, propylphenyl and isopropylphenyl, by coupling of amino acids, at least one of said amino acids bearing said aryloxycarbonyl group attached to a nitrogen atom of an amino functional group, wherein, after incorporation into a peptide fragment of said amino acid bearing said aryloxycarbonyl group, the intermediate peptide synthesis is continued by coupling said peptide fragment with amino acids in a conventional manner, the aryloxycarbonyl group acting as a stable blocking group of the amino functional group to which it is attached during the synthesis of said intermediate peptide; and b) reacting said intermediate peptide with a compound of general formula R1R2NH in which R1 and R2 represent, independently of each other, hydrogen atoms, alkyl, cycloalkyl or aralkyl radicals, containing at most 12 carbon atoms, or in which R1 and R2 together form an alicyclic radical containing 3 to 6 carbon atoms, to produce said peptide containing at least one N-carbamoyl functional group, the aryloxycarbonyl group acting as a specific intermediate for the desired N-carbamoyl functional group.
 10. The method according to claim 9, wherein R3 is a phenyl or p-tolyl group.
 11. The method according to claim 9, wherein R3 is phenyl.
 12. The method according to claim 9, wherein the compound R1R2NH is used in a quantity 2 to 100 times greater than a stoichiometric quantity.
 13. The method according to claim 9, wherein the intermediate peptide is synthesized starting with the N.sup.ω -aryloxycarbonyl derivative of an α, ω diamino acid.
 14. The method according to claim 9 wherein the peptides produced are selected from the group consisting of peptides containing L, D, or DL norcitrulline, citrulline or homocitrulline residues from corresponding L, D, or DL 2,4-diaminobutyric acid, ornithine, and lysine respective precursor amino acids. 